The application of hematopoietic stem cell gene therapy to sickle cell disease (SCD) may one day offer the chance of cure to patients with this disorder. Although significant progress has been made towards this goal, many obstacles remain. The major obstacle to the gene therapy of SCD at this stage is the very low efficiency of gene transfer into hematopoietic stem cells. Current approaches to the gene therapy of SCD aim at inserting a normal globin gene in the genome of the hematopoietic stem cell rather than replacing the mutant gene with a normal gene. Such gene "addition" would result in excess synthesis of normal globin chains in a cell with a full complement of mutant chains. The effects of this manipulation on the overall hemoglobin composition of the red cell and its propensity to sickle are unknown. This is a very difficult situation then a sickle cell patient with a high level of fetal hemoglobin or a sickle beta-thalassemia patient with a high level of hemoglobin a where the normal or beta-globin chains replace some of the mutant sickle globin chains in the red cell. As a result of the low efficiency of gene transfer to hematopoietic stem cells, it is not possible to determine the effects of such gene transfer in an in vivo setting. We propose to develop an in vitro system to assess the effects of the transfer of normal globin genes into erythroid progenitors of patients with SCD on the phenotype of the transduced cells. The recent development of in vitro methods for the expansion and differentiation of phenotype of the transduced cells. The recent development of in vitro methods for the expansion and differentiation of erythroid in a two-phase liquid culture system is crucial for the success for such a system. This has made it possible to retrovirally transduce and select erythroid progenitors that incorporate the transferred globin genes and expand and differentiate them, all in an in vitro culture system. The effects of gene transfer on the hemoglobin phenotype and the sickling potential of transduced cells from patients with sickling disorders can then be analyzed by established in vitro assays of red blood sickling. We will analyze the effects of gene transfer on the phenotype of cells from patients with a variety of sickling disorders including SS, SC, Sbeta- thalassemia, all in the presence and absence of interacting alpha- thalassemia, this experimental system is designed to provide the proof of principle that this type of gene transfer may be effective in preventing sickling and its complications. We also hope it will serve as a useful pre-clinical test for comparing the effectiveness of different gene transfer vector in a safe and relatively inexpensive in vitro setting.